This invention relates in general to capillary electrophoretic systems and in particular to an improved capillary tube with a structure that permits electrical contact through ionic movement and to a capillary electrophoretic system employing such a capillary tube.
Capillary zone electrophoresis (CZE) in small capillaries has proven useful as an efficient method for the separation of solutes. An electric field is applied between the two ends of a capillary tube into which an electrolyte containing the solutes is introduced. The electric field causes the electrolyte to flow through the tube. Some solutes will have higher electrokinetic mobilities than other solutes so that the sample components are resolved into zones in the capillary tube during the flow of the electrolytes through the capillary.
CZE is advantageous because it requires only very small sample volumes, such as the contents of a cell or cellular subcompartments. For these and other reasons, CZE has shown great promise as a separation and detection technique.
A number of CZE detection schemes have been used. These schemes include optical detection based on optical properties of sample components. Other CZE detection schemes include conductivity detection and electrochemical detection. In conductivity detection, a conductivity detector detects the change in conductance between two points as different zones move through the detection region. In electrochemical detection, electroactive sample components are detected when they alter the voltage across or the current between two points near the detector.
In the electrophoresis process, a high voltage of the order of several tens of thousands of volts is applied between the two ends of a capillary tube in order to move the electrolyte and samples through the tube in a reasonable time and with reasonable resolution. Such high voltage used may create high electric fields at a detecting or measuring device which may introduce unacceptable noise or otherwise interfere with the measurement of such a device and which may, in the extreme, cause damage to the circuitry in the device. Therefore in order for conductivity or electrochemical detectors to function properly in an electrophoresis process, the electrolyte and sample at or near the location of detection should be either grounded or fixed at a low voltage which will not interfere with the measurements of the detectors.
In conductivity detection, the voltage across and current between two selected points are measured so that it is necessary to introduce two electrodes, one at each selected point. It is therefore desirable to provide electrophoretic devices that permit electrodes to be introduced at or near the points of detection or which otherwise provides means to at least maintain such points at ground or a low voltage.
In a conventional capillary electrophoresis system, each end of the capillary is dipped into a reservoir. A high voltage is then applied between the two reservoirs. Typically, a high voltage is applied to one reservoir and the other reservoir is grounded. The two reservoirs each contains an electrolyte buffer. Such a conventional system is described in "Capillary Electrophoresis" by Gordon, Huang, Pentoney, Jr. and Zare, reprint series from Science, Oct. 14, 1988, Vol. 242, pp. 224-228.
In the conventional capillary electrophoresis system, the reservoir connected to one of the two ends of the capillary is grounded so that conductivity and electrochemical detections may be performed near the tube at the location close to such reservoir. Such a conventional system is disadvantageous because any sample components collected will be diluted by the electrolyte buffer in the reservoir and the system does not permit continuous collection of sample components. Furthermore, since the detector itself in conductivity or electrochemical detection cannot be placed directly in the electrolyte buffer, but must be placed at a location at least a short distance removed from the reservoir, the sample components actually detected by the detector is not grounded or fixed at a set low voltage. This may result in serious inaccuracies in the measurements.
In view of the above disadvantages of the conventional electrophoresis system, various solutions have been proposed. One alternative electrophoresis system is disclosed in "Improved Electrospray Ionization Interface for Capillary Zone Electrophoresis-Mass Spectrometry" by Smith et al., Anal. Chem. 1988, 60, 1948-1952. In the paper, Smith et al. described a previous ionization interface where a metal electrode coated on the outlet of the capillary is used to ground the electrolyte and sample. As Smith et al. admitted in the paper, the process of depositing metal at a capillary terminus was time consuming and the deposited metal was slowly eroded by electrochemical processes and required replacement after several days of operation. In many cases, electrochemical reaction at the electrode lead to the evolution of gas bubbles which were extremely detrimental to the operation of the electrophoretic separation. This scheme also suffers from irreproducibility caused by electrochemical reaction at the grounding electrode.
In the same paper, Smith et al. also described another ionization interface utilizing a liquid sheath electrode. The actual CZE electrical contact is made to the low-voltage end of the capillary with a thin sheath of liquid that flows over the outside surface of the capillary. In such a scheme, the CZE effluent avoids contact with any metal surfaces and is isolated from loss by electrochemical reactions. The system is difficult to construct. With such a scheme, sample components collected will be diluted by the buffer liquid in the liquid sheath.
In "Capillary zone electrophoresis with electrochemical detection", Anal. Chem. 1987, 59, 1762-1766 and "Amperometric Detection of Catechols in Capillary Zone Electrophoresis with Normal and Micellar Solutions", Anal. Chem. 1988, 60, 258-263, Wallingford and Ewing disclosed a conductive joint made near the low-voltage end of the capillary by fracturing the capillary at such a point and encasing the fracture in a porous glass capillary whose inside diameter was large enough for encasing the sample holding capillary. The porous glass capillary permitted ionic movement through the fracture between the sample and an outside reservoir for grounding or for setting the electrolyte and sample at the fracture point to ground or a low voltage. Again this scheme is difficult to construct. Furthermore, the system had a dead volume (extra volume) at the fracture point which decreased sensitivity and resolution.
Because none of the above described schemes is entirely satisfactory, it is desirable to provide an electrophoretic device in which the above-described difficulties are alleviated.